Use of defibrinated blood for manufacture of a hemoglobin-based oxygen carrier

ABSTRACT

Red blood cells are purified by defibrinating whole blood and then filtering the defibrinated whole blood, whereby at least a portion of a plasma component is separated from the red blood cells to form a suspension of red blood cells, thereby purifying the red blood cells. Whole blood is defibrinated by, for example, using a chemical coagulating agent or mechanical agitation. Separation of the plasma component from red blood cells can be completed by, for example, diafiltration. The suspension of red blood cells can then be employed to produce a hemoglobin-based oxygen carrier.

RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.09/795,821, filed Feb. 28, 2001, now U.S. Pat. No. 6,518,010. The entireteaching of the above application is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The development of hemoglobin-based oxygen carriers has focused onoxygen delivery for use in medical therapies such as transfusions andthe production of blood products. Hemoglobin-based oxygen carriers canbe used to prevent or treat hypoxia resulting from blood loss (e.g, fromacute hemorrhage or during surgical operations), from anemia (e.g.,pernicious anemia or sickle cell anemia), or from shock (e.g, volumedeficiency shock, anaphylactic shock, septic shock or allergic shock).

Existing hemoglobin-based oxygen carriers include perfluorochemicals,synthesized hemoglobin analogues, liposome-encapsulated hemoglobin,chemically-modified hemoglobin, and hemoglobin-based oxygen carriers inwhich the hemoglobin molecules are crosslinked. Preparation ofhemoglobin-based oxygen carriers includes several purification steps.Among the components that must be removed from collected blood isfibrinogen, which is a soluble protein that is converted into fibrin bythe action of thrombin during clotting. Current techniques forprocessing blood often include addition of chemical agents, such assodium citrate, to prevent coagulation. However, additional techniqueswhich might, for example, reduce the expense of processing, withoutdiminishing other qualities, such as ultimate product purity, aresought.

SUMMARY OF THE INVENTION

The present invention relates to the use of defibrinated blood forpurifying red blood cells, preparing a hemoglobin solution, andpreparing a hemoglobin-based oxygen carrier. Chemical clotting agents(such as collagen) and mechanical agitation (such as stirring) aremethods used to defibrinate blood. Subsequent cell washing removesplasma proteins that may lead to incompatibility between donor andrecipient blood.

In one embodiment, the method for purifying red blood cells includesdefibrinating whole blood, the whole blood including red blood cells anda plasma component. Subsequently, the whole blood is filtered to purifythe red blood cells and thereby form a red blood cell suspension.

In an embodiment of the method for preparing a hemoglobin solution,whole blood is defibrinated. Red blood cells are separated from thewhole blood, and hemoglobin molecules are isolated from the red bloodcells to form thereby a hemoglobin solution.

In one embodiment of the method to prepare a hemoglobin-based oxygencarrier, whole blood is defibrinated. Red blood cells are separated fromthe whole blood. Hemoglobin molecules are isolated and stabilized toform the hemoglobin-based oxygen carrier.

The advantages of this invention are numerous. One advantage is that theinvention obviates the need for an anticoagulant solution to be mixedwith whole blood (human, bovine, mammalian). Adding an anticoagulantinvolves manpower and capital for the processes of preparation of highpurity water mixing solutions, preparation of citrated collectioncontainers, collection, mixing, and purification. In addition, whenshipping blood, generally it is easier to defibrinate blood than it isto build facilities for addition of an anticoagulant at the shipper'slocation.

BRIEF DESCRIPTION OF THE DRAWINGS

The FIGURE is a schematic of an embodiment of apparatus suitable forconducting the method of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The foregoing and other objects, features and advantages of theinvention will be made more apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawing. The drawing is not necessarily to scale,emphasis instead being placed upon illustrating the principles of theinvention.

Generally, the invention is a method for purifying blood to form a redblood cell (RBC) suspension, to isolate a hemoglobin solution, and tomanufacture a hemoglobin-based oxygen carrier. The method includesdefibrinating whole blood. For the purpose of describing the invention,whole blood is considered to be comprised of red blood cells and plasmacomponents.

Referring to the FIGURE, shown therein is one embodiment of apparatus 10suitable for practicing the method of the invention. Whole blood iscollected in vessel 12. Whole blood suitable for use in the inventioncan be freshly collected or from otherwise outdated sources, such asexpired human blood from a blood bank. Further, the whole blood can havebeen maintained in a frozen and/or a liquid state, although it ispreferred that the whole blood has not been frozen prior to use in thismethod. Examples of suitable whole blood sources include human, bovine,ovine, porcine, other vertebrates and transgenically-producedhemoglobin, such as the transgenic Hb described in BIO/TECHNOLOGY, 12:55-59 (1994), the teachings of which are incorporated herein byreference in their entirety. The blood can be collected from live orfreshly slaughtered animal donors. One method for collecting bovinewhole blood is described in U.S. Pat. Nos. 5,084,558 and 5,296,465,issued to Rausch et al., the teachings of which are incorporated byreference in their entirety.

The whole blood is defibrinated in vessel 12 by a suitable method.Defibrinating the blood sets off the clotting cascade to removeartificially the fibrin molecules involved in the formation of bloodclots. Defibrination can be induced by chemical or mechanical means.Chemical coagulating agents are defined herein as substances that induceclotting. For example, collagen induces coagulation so that when thereis an external wound, a fibrin clot will stop blood from flowing.Artificially exposing blood to collagen will cause the formation offibrin clots, which can be removed to produce defibrinated blood.

In one embodiment, the blood is defibrinated by exposure to acoagulating agent. Examples of coagulating agents are collagen, tissueextract, tissue factor, tissue thromboplastin, anionic phospholipid,calcium, negatively charged materials (e.g., glass, kaolin, somesynthetic plastics, fabrics). A preferred clotting agent is collagen.

The whole blood is exposed to the clotting agent for a period of timesufficient to cause essentially all fibrin in the blood to be convertedinto a fibrin clot. The appropriate time is determined by the point atwhich fibrin molecules apparently stop polymerizing. Chemicaldefibrination, defined herein as defibrination that is induced byexposure to a chemical coagulating agent, is conducted at a suitabletemperature, preferably a temperature in a range of between about 4° C.and about 40° C.

In another embodiment, mechanical agitation, such as stirring, also hasthe effect of initiating the clotting cascade. After stirring untilfibrin polymerization apparently ceases, it is possible to remove theaccumulated fibrin to complete defibrination. Mechanical defibrination,defined herein as defibrination induced by agitating the blood solution,is conducted at a suitable temperature, and preferably at a temperaturein a range of between about 4° C. and about 40° C.

Fibrin is then removed from the whole blood by a suitable means. Anexample of a suitable means is by directing the whole blood, includingthe fibrin, from vessel 12, through line 14 and strainer 16. A 60 meshscreen is an example of a suitable strainer. Fibrin is collected atstrainer 16 and the remainder of the whole blood is directed to vessel18. Optionally, or alternatively to the use of a strainer, cheeseclothor polypropylene filters can be employed to remove large debris,including fibrin.

As shown in the FIGURE, whole blood is directed from vessel 18 throughline 20 by pump 22 and through first filter 24 and second filter 26 tovessel 28. In one embodiment, first filter 24 and second filter 26 arepolypropylene filters. In a particularly preferred embodiment, firstfilter 24 has a permeability of about 800 μm, and second filter 26 has apermeability of about 50 μm. Removal of essentially all of the fibrin byfirst filter 24 and second filter 26 completes the defibrination step.

The whole blood is maintained at a suitable temperature in vessel 28.Preferably, the whole blood is maintained at a temperature in a range ofbetween about 4° C. and about 15° C. The temperature of whole blood invessel 28 is maintained by recirculation of a suitable medium, such asethylene glycol, through jacket 30 at vessel 28. Recirculation of mediumthrough jacket 30 is maintained by line 32, reservoir 34, pumps 36, 38and chiller, or refrigeration unit, 40.

Thereafter, the whole blood is filtered, whereby at least a portion ofthe plasma component is separated from the red blood cells to form a redblood cell suspension, thereby purifying the red blood cells.Preferably, the whole blood is filtered by diafiltration.

In one embodiment, diafiltration is conducted by diverting whole bloodfrom vessel 28 through line 42 and pump 44 to diafiltration module 46.Diafiltration module 46 includes inlet 48, retentate outlet 50 andpermeate outlet 52. Membrane 54 partitions retentate portion 56 ofdiafiltrate module 46 from permeate portion 58 of diafiltrate module 46.Preferably, membrane 54 has a permeability limit in a range of betweenabout 0.01 μm and about 5 μm.

A portion of the plasma component of whole blood in diafiltrate module46 passes across membrane 54 from retentate portion 56 to permeateportion 58, thereby purifying red blood cells at retentate portion 56.Purified red blood cells are directed through retentate outlet 50 andline 60 back to vessel 28. Purified blood can be collected from vessel28 through valve 62 to line 64 for further processing. Plasma thatpermeates membrane 54 can be directed from permeate portion 58 ofdiafiltration module 46 through line 66 and collected from vessel 68.Blood recirculating through vessel 28 and diafiltrate module 46 can besampled at sampling ports (not shown) in line 42 or line 60.

Preferably, prior to filtering whole blood to remove at least a portionof the plasma component, a liquid is added to the whole blood in vessel28 from vessel 70 and line 72 to dilute its concentration. In oneembodiment, the whole blood is diluted to a concentration in a range ofbetween about 25% and about 75% of its initial concentration (beforedilution), by volume. Concentration then can reduce the volume back tothe original concentration or more. Generally, the process of adding aliquid to the whole blood and then removing at least a portion of theliquid, is referred to as “cell washing.”

In one embodiment, cell washing includes the processes of dilution anddiafiltration in a continuous filtration operation; a saline/citratesolution is added to the filter retentate to maintain a constant volumein the recirculation tank. The result is a reduction in theconcentration of microfiltration membrane-permeable species (includingmembrane-permeable plasma proteins). Subsequent reconcentration of thediluted blood solution back to the original volume produces a purifiedblood solution.

In a preferred embodiment, the blood solution is washed by diafiltrationor by a combination of discrete dilution and concentration steps with atleast one solution, such as an isotonic solution, to separate red bloodcells from extracellular plasma proteins, such as serum albumins orantibodies (e.g., immunoglobulins (IgG)). Preferably, the isotonicsolution includes an ionic solute or is aqueous. It is understood thatthe red blood cells can be washed in a batch or continuous feed mode.

Acceptable isotonic solutions are known in the art and includesolutions, such as a citrate/saline solution, having a pH and osmolaritywhich does not rupture the cell membranes of red blood cells and whichdisplaces the plasma portion of the whole blood. The blood may bediluted to a concentration in the range between about 25% and 75% of theoriginal concentration. A preferred isotonic solution has a neutral pHand an osmolarity-between about 285-315 mOsm. In a preferred embodiment,the isotonic solution is composed of an aqueous solution of sodiumcitrate dihydrate (6.0 g/l) and of sodium chloride (8.0 g/l).

In one method, the whole blood is diafiltered across a membrane having apermeability limit in the range of between 0.2 μm and about 2.0 μm.Alternate suitable diafilters include microporous membranes with poresizes that will separate RBCs from substantially smaller blood solutioncomponents, such as a 0.1 μm to 0.5 μm filter (e.g., a 0.2 μm hollowfiber filter, Microgon Krosflo II microfiltration cartridge). Duringcell washing, fluid components of the blood solution, such as plasma, orcomponents which are significantly smaller in diameter than RBCs passthrough the walls of the diafilter in the filtrate. Erythrocytes,platelets and larger bodies of the blood solution, such as white bloodcells, are retained and mixed with isotonic solution, which is addedcontinuously or batch-wise to form a dialyzed blood solution.

Concurrently, a filtered isotonic solution is added continuously (or inbatches) as makeup to maintain volume of filtrate to compensate for theportion of the solution lost across the diafilter. In a more preferredembodiment, the volume of blood solution in the diafiltration tank isinitially diluted by the addition of a volume of a filtered isotonicsolution to the diafiltration tank. Preferably, the volume of isotonicsolution added is about equal to the initial volume of the bloodsolution.

In an alternate embodiment, the blood is washed through a series ofsequential (or reverse sequential) dilution and concentration steps,wherein the blood solution is diluted by adding at least one isotonicsolution, and is concentrated by flowing across a filter, therebyforming a dialyzed blood solution.

Cell washing generally is considered to be complete when the level ofplasma proteins contaminating the red blood cells has been substantiallyreduced (typically at least about 90%). Additional washing may furtherseparate extracellular plasma proteins from the RBCs. For instance,diafiltration with six volumes of isotonic solution may be sufficient toremove at least about 99% of IgG from the blood solution.

Potential foulants of the membrane could cause problems with washing,such as slow manufacturing runs, which may be minimized by using newmembranes for each run of washing. However, it is still possible to makean effective hemoglobin-based oxygen carrier, despite potential membranefoulants. Small fibrin molecules can be problematic and may foul thefilter if they accumulate on the surface of a membrane with apermeability of 0.1 to 5 μm and thus block the pores. A narrower rangein which the foulants can be problematic is 0.2 to 0.4 μm. Defibrinating(mechanical, chemical, any kind) could cause red blood cell lysing. Redblood cells, white blood cells, or platelets that have broken open mightstick to the filter.

In another embodiment of the invention, it is possible to defibrinateblood that has already been citrated by saturating the citrated bloodwith a divalent cation, and then defibrinating the solution, similar tothe means by which uncitrated blood would be processed. The preferreddivalent cation is calcium.

To prepare a hemoglobin blood solution, the purified blood sample can befurther processed to isolate the hemoglobin molecules. The resultingdialyzed blood solution is exposed to means for separating red bloodcells in the dialyzed blood solution from white blood cells andplatelets, such as by centrifugation. It is understood that othermethods generally known in the art for separating red blood cells fromother blood components can be employed. For example, one embodiment ofthe invention separates red blood cells by sedimentation, wherein theseparation method does not rupture the cell membranes of a significantamount of the RBCs, such as less than about 30% of the RBCs, prior tored blood cell separation from the other blood components.

Following purification of the red blood cells, the RBCs are lysed,resulting in the production of a hemoglobin (Hb) solution. Methods oflysis include mechanical lysis, chemical lysis, hypotonic lysis or otherknown lysis methods which release hemoglobin without significantlydamaging the ability of the Hb to transport and release oxygen.

Following lysis, the lysed red blood cell phase is then ultrafiltered toremove larger cell debris, such as proteins with a molecular weightabove about 100,000 Daltons. The hemoglobin is then separated from thenon-Hb components of the filtrate.

Methods of ultrafiltration and methods of separating Hb from non-Hbcomponents by pH gradients and chromatography are further described inU.S. Pat. No. 5,691,452, filed Jun. 7, 1995, which is incorporated byreference in its entirety.

The Hb eluate is then preferably deoxygenated prior to polymerization toform a deoxygenated Hb solution (hereinafter deoxy-Hb) for furtherprocessing into a hemoglobin-based oxygen carrier. In a preferredembodiment, deoxygenation substantially deoxygenates the Hb withoutsignificantly reducing the ability of the Hb in the Hb eluate totransport and release oxygen, such as would occur from formation ofoxidized hemoglobin (metHb). Alternatively, the hemoglobin solution maybe deoxygenated by chemical scavenging with a reducing agent selectedfrom the group consisting of N-acetyl-L-cysteine (NAC), cysteine, sodiumdithionite or ascorbate.

The method of deoxygenation is further described in U.S. Pat. No.5,895,810, filed Jun. 7, 1995, which is incorporated herein by referencein its entirety.

The deoxygenated hemoglobin solution can be further processed into ahemoglobin-based oxygen carrier. As defined herein, a “hemoglobin-basedoxygen carrier” is a hemoglobin-based composition suitable for use inhumans, mammals, and other vertebrates, which is capable of transportingand transferring oxygen to vital organs and tissues, at least, and canmaintain sufficient intravascular oncotic pressure, wherein thehemoglobin has been isolated from red blood cells. A vertebrate is asclassically defined, including humans, or any other vertebrate animalswhich uses blood in a circulatory system to transfer oxygen to tissue.Additionally, the definition of circulatory system is as classicallydefined, consisting of the heart, arteries, veins and microcirculationincluding smaller vascular structures such as capillaries.

“Stable polymerized hemoglobin”, as defined herein, is a component of ahemoglobin-based oxygen carrier composition which does not substantiallyincrease or decrease in molecular weight distribution and/or inmethemoglobin content during storage periods at suitable storagetemperatures for periods of about two years or more. Suitable storagetemperatures for storage of one year or more are between about 0° C. andabout 40° C. The preferred storage temperature range is between about 0°C. and about 25° C.

A suitable low oxygen environment, or an environment that issubstantially oxygen-free, is defined as the cumulative amount of oxygenin contact with the hemoglobin-based oxygen carrier, over a storageperiod of at least about two months, preferably at least about one year,or more preferably at least about two years, which will result in amethemoglobin concentration of less than about 15% by weight in thehemoglobin-based oxygen carrier. The cumulative amount of oxygenincludes the original oxygen content of the hemoglobin-based oxygencarrier and packaging in addition to the oxygen resulting fromoxygen-leakage into the hemoglobin-based oxygen carrier packaging.

Throughout this method, from RBC collection until hemoglobinpolymerization, blood solution, RBCs and hemoglobin are maintained underconditions sufficient to minimize microbial growth, or bioburden, suchas maintaining temperature at less than about 20° C. and above 0° C.Preferably, temperature is maintained at a temperature of about 15° C.or less. More preferably, the temperature is maintained at 10±2° C.

In this method, portions of the components for the process of preparinga stable polymerized hemoglobin-based oxygen carrier are sufficientlysanitized to produce a sterile product. Sterile is as defined in theart, specifically, in the United States Pharmacopeia requirements forsterility provided in USP XXII, Section 71, pages 1483-1488. Further,portions of components that are exposed to the process stream, areusually fabricated or clad with a material that will not react with orcontaminate the process stream. Such materials can include stainlesssteel and other steel alloys, such as Hasteloy.

In one embodiment, polymerization results from intramolecularcross-linking of Hb. The amount of a sulfhydryl compound mixed with thedeoxy-Hb is high enough to increase intramolecular cross-linking of Hbduring polymerization and low enough not to significantly decreaseintermolecular cross-linking of Hb molecules, due to a high ionicstrength. Typically, about one mole of sulfhydryl functional groups(—SH) are needed to oxidation-stabilize between about 0.25 moles toabout 5 moles of deoxy-Hb.

Optionally, prior to polymerizing the oxidation-stabilized deoxy-Hb, anappropriate amount of water is added to the polymerization reactor. Inone embodiment, an appropriate amount of water is that amount whichwould result in a solution with a concentration of about 10 to about 100g/l Hb when the oxidation-stabilized deoxy-Hb is added to thepolymerization reactor. Preferably, the water is oxygen-depleted.

The temperature of the oxidation-stabilized deoxy-Hb solution in thepolymerization reactor is raised to a temperature to optimizepolymerization of the oxidation-stabilized deoxy-Hb when contacted witha cross-linking agent. Typically, the temperature of theoxidation-stabilized deoxy-Hb is about 25 to about 45° C., andpreferably about 41 to about 43° C. throughout polymerization. Anexample of an acceptable heat transfer means for heating thepolymerization reactor is a jacketed heating system which is heated bydirecting hot ethylene glycol through the jacket.

The oxidation-stabilized deoxy-Hb is then exposed to a suitablecross-linking agent at a temperature sufficient to polymerize theoxidation-stabilized deoxy-Hb to form a solution of polymerizedhemoglobin (poly(Hb)) over a period of about 2 hours to about 6 hours. Asuitable amount of a cross-linking agent is that amount which willpermit intramolecular cross-linking to stabilize the Hb and alsointermolecular cross-linking to form polymers of Hb, to thereby increaseintravascular retention. Typically, a suitable amount of a cross-linkingagent is that amount wherein the molar ratio of cross-linking agent toHb is in excess of about 2:1. Preferably, the molar ratio ofcross-linking agent to Hb is between about 20:1 to 40:1.

Examples of suitable cross-linking agents include polyfunctional agentsthat will cross-link Hb proteins, such as glutaraldehyde,succindialdehyde, activated forms of polyoxyethylene and dextran,α-hydroxy aldehydes, such as glycolaldehyde,N-maleimido-6-aminocaproyl-(2′-nitro,4′-sulfonic acid)-phenyl ester,m-maleimidobenzoic acid-N-hydroxysuccinimide ester, succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate, sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester,m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-succinimidyl(4-iodoacetyl)aminobenzoate,sulfosuccinimidyl(4-iodoacetyl)aminobenzoate, succinimidyl4-(p-maleimidophenyl)butyrate, sulfosuccinimidyl4-(p-maleimidophenyl)butyrate,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,N,N′-phenylene dimaleimide, and compounds belonging to the bis-imidateclass, the acyl diazide class or the aryl dihalide class, among others.

Poly(Hb) is defined as having significant intramolecular cross-linkingif a substantial portion (e.g., at least about 50%) of the Hb moleculesare chemically bound in the poly(Hb), and only a small amount, such asless than about 15%, are contained within high molecular weight poly(Hb)chains. High molecular weight poly(Hb) molecules have a molecule weight,for example, above about 500,000 Daltons.

In a preferred embodiment, glutaraldehyde is used as the cross-linkingagent. Typically, about 10 to about 70 grams of glutaraldehyde are usedper kilogram of oxidation-stabilized deoxy-Hb. More preferably,glutaraldehyde is added over a period of five hours until approximately29-31 grams of glutaraldehyde are added for each kilogram ofoxidation-stabilized deoxy-Hb.

Wherein the cross-linking agent used is not an aldehyde, the poly(Hb)formed is generally a stable poly(Hb). Wherein the cross-linking agentused is an aldehyde, the poly(Hb) formed is generally not stable untilmixed with a suitable reducing agent to reduce less stable bonds in thepoly(Hb) to form more stable bonds. Examples of suitable reducing agentsinclude sodium borohydride, sodium cyanoborohydride, sodium dithionite,trimethylamine, t-butylamine, morpholine borane and pyridine borane. Thepoly(Hb) solution is optionally concentrated by ultrafiltration untilthe concentration of the poly(Hb) solution is increased to between about75 and about 85 g/l. For example, a suitable ultrafilter is a 30,000Dalton filter (e.g., Millipore Helicon Cat # CDUF050LT; Amicon Cat #540430).

The pH of the poly(Hb) solution is then adjusted to the alkaline pHrange to preserve the reducing agent and to prevent hydrogen gasformation, which can denature Hb during the subsequent reduction. Thepoly(Hb) is typically purified to remove non-polymerized hemoglobin.This can be accomplished by dialfiltration or hydroxyapatitechromatography (see, e.g. U.S. Pat. No. 5,691,453, filed Jun. 7, 1995,which is incorporated herein by reference in its entirety). Following pHadjustment, at least one reducing agent, preferably a sodium borohydridesolution, is added to the polymerization step typically through thedeoxygenation loop. The pH and electrolytes of the stable poly(Hb) canthen be restored to physiologic levels to form a stable polymerizedhemoglobin-based oxygen carrier, by diafiltering the stable poly(Hb)with a diafiltration solution having a suitable pH and physiologicelectrolyte levels.

Suitable methods of cross-linking hemoglobin and preserving thehemoglobin-based oxygen carrier are discussed in detail in U.S. Pat. No.5,691,452, issued Nov. 25, 1997, which is incorporated herein byreference in its entirety.

Vertebrates that can receive the hemoglobin-based oxygen carrier, formedby the methods of the invention, include mammals, such as humans,non-human primates, dogs, cats, rats, horses, or sheep. Further,vertebrates, that can receive said hemoglobin-based oxygen carrier,include fetuses (prenatal vertebrate), post-natal vertebrates, orvertebrates at time of birth.

A hemoglobin-based oxygen carrier of the present invention can beadministered into the circulatory system by injecting thehemoglobin-based oxygen carrier directly and/or indirectly into thecirculatory system of the vertebrate, by one or more injection methods.Examples of direct injection methods include intravascular injections,such as intravenous and intra-arterial injections, and intracardiacinjections. Examples of indirect injection methods includeintraperitoneal injections, subcutaneous injections, such that thehemoglobin-based oxygen carrier will be transported by the lymph systeminto the circulatory system or injections into the bone marrow by meansof a trocar or catheter. Preferably, the hemoglobin-based oxygen carrieris administered intravenously.

The vertebrate being treated can be normovolemic, hypervolemic orhypovolemic prior to, during, and/or after infusion of thehemoglobin-based oxygen carrier. The hemoglobin-based oxygen carrier canbe directed into the circulatory system by methods such as top loadingand by exchange methods.

A hemoglobin-based oxygen carrier can be administered therapeutically,to treat hypoxic tissue within a vertebrate resulting from manydifferent causes including anemia, shock, and reduced RBC flow in aportion of, or throughout, the circulatory system. Further, thehemoglobin-based oxygen carrier can be administered prophylactically toprevent oxygen-depletion of tissue within a vertebrate, which couldresult from a possible or expected reduction in RBC flow to a tissue orthroughout the circulatory system of the vertebrate. Further discussionof the administration of hemoglobin to therapeutically orprophylactically treat hypoxia, particularly from a partial arterialobstruction or from a partial blockage in microcirculation, and thedosages used therein, is provided in U.S. Pat. No. 5,854,209, filed Mar.23, 1995, which is incorporated herein by reference in its entirety.

Typically, a suitable dose, or combination of doses of hemoglobin-basedoxygen carrier, is an amount which when contained within the bloodplasma will result in a total hemoglobin concentration in thevertebrate's blood between about 0.1 to about 10 grams Hb/dl, or more,if required to make up for large volume blood losses.

The invention will now be further and specifically described by thefollowing examples.

EXEMPLIFICATION EXAMPLE 1 Bench Scale Experiment

The bench-scale experiments were performed in the apparatus shown in theFIGURE. The defibrinated blood sample used in the bench scale experimentwas defibrinated by exposure to collagen. Initially, whole blood isdiluted approximately 1:1 with isotonic citrate saline buffer. Thediluted blood was then concentrated back to produce a Hb level of 10.5g/dl (approximately a two-fold concentration). The process volume forthe diafiltration was 200 ml, therefore approximately 200 ml buffer wasadded to approximately 200 ml whole blood followed by concentration backto its original volume. This produced approximately 200 ml of membranepermeate. The 200 ml whole blood at a Hb concentration of 10.5 g/dL wasthen diafiltered against citrate/saline buffer. The time to collect 400mls permeate volume (2 retentate volumes) was used as a point ofcomparison for the citrated blood and the defibrinated blood. The timeincluded the time to concentrate the diluted blood back to its originalvolume (200 ml) and the time to perform the first diafiltration volume(200 ml). The longer the time, the slower the process. Table 1summarizes the results.

TABLE 1 Time to Collect 400 ml Animal Number Whole Blood Sample Permeate(Hr: Min: Sec) 1 Citrated 0:25:08 1 Defibrinated 0:51:06 2 Citrated0:24:47 2 Defibrinated 0:25:15 3 Citrated 0:25:08 3 Defibrinated 0:24:284 Citrated 0:30:18 4 Defibrinated 0:15:57

As can be seen from Table 1, the time required to collect 400 ml ofpermeate was between about fifteen minutes and an hour.

EXAMPLE 2 Pilot Scale Experiment

The pilot-scale experiments were performed in the apparatus shown in theFIGURE. The defibrinated blood sample used in the pilot-scale experimentwas defibrinated by mechanical agitation. Again, the whole blood isdiluted with isotonic citrate saline solution and concentrated, butbecause of the large volume required for processing in the pilot scalesystem, the initial whole blood was diluted with a greater than 1:1ratio of citrate/saline buffer to whole blood. The Hb concentration ofthe blood during diafiltration is less than 10.5 g/dl (approximately atwo-fold concentration). After concentration back to the minimum processvolume of the system (approximately 4.5 L), the blood was diafilteredfor 5 diafiltration volumes. As in the bench scale experiment, a longertime indicates a slower process. Table 2 summarizes the results.

TABLE 2 Experiment Processing Time to Collect 5 Number Whole BloodSample Diafiltration Volumes (minutes) 1 Citrated (Control) 91Defibrinated 28 2 Citrated (Control) 83 Defibrinated 150

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all other suchequivalents are intended to be encompassed by the following claims.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for preparing a hemoglobin solution, comprising the stepsof: a) defibrinating whole blood, wherein the whole blood comprises redblood cells and a plasma component; b) filtering the defibrinated wholeblood by diafiltration across a membrane having a permeability limit ina range of between about 0.01 μm and about 5 μm, whereby at least aportion of the plasma component is separated from the red blood cells toform a red blood cell suspension; and thereafter c) releasing hemoglobinmolecules from the red blood cells of the red blood cell suspension bylysing the red blood cells of the red blood cell suspension andisolating the hemoglobin molecules by centrifuging or filtering thelysed red blood cell suspension; whereby a hemoglobin solution isformed.
 2. The method of claim 1, wherein centrifuging the red bloodcell suspension causes at least a portion of the red blood cells tolyse, thereby releasing hemoglobin molecules.
 3. The method of claim 2,wherein the released hemoglobin is isolated by centrifugation.
 4. Themethod of claim 2, wherein the released hemoglobin is isolated byfiltration.
 5. The method of claim 1, wherein the red blood cells arelysed by centrifuging the red blood cell suspension cells that have beenseparated in step b).
 6. The method of claim 1, wherein the red bloodcells are lysed hypotonically.
 7. The method of claim 1, furthercomprising the step of deoxygenating the hemoglobin solution.
 8. Themethod of claim 7, wherein the content of an oxyhemoglobin component ofthe hemoglobin solution is reduced to less than about 20%.
 9. The methodof claim 7, wherein the oxyhemoglobin component of the hemoglobinsolution is reduced to less than about 10%.
 10. The method of claim 7,wherein the hemoglobin solution is deoxygenated by chemical scavengingwith a reducing agent.
 11. The method of claim 10, wherein the reducingagent is selected from the group consisting of N-acetyl-L-cysteine(NAC), cysteine, sodium dithionite or ascorbate.